Molecular and chemical evaluation of patulin production of Aspergillus and Penicillium-like species isolated from Hungarian apples.

Mycotoxins are secondary fungal metabolites harmful to humans and animals. Patulin (PAT) is a toxin found in different food products but especially in apples and their derivative products. The most common fungi producers of this compound are Aspergillus clavatus and Penicillium expansum. The production of patulin, as other mycotoxins, can be impacted by diverse phenomena such as water and nutrient availability, UV exposure, and the presence of antagonistic organisms. Consequently, gaining a comprehensive understanding of climate and environmental conditions is a crucial step in combating patulin contamination. In this study, moulds were isolated from 40 apple samples collected from seven locations across Hungary: Csenger, Damak, Pallag, Lövopetri, Nagykálló, and Újfehértó. A total of 183 moulds were morphologically identified, with 67 isolates belonging to the Alternaria, 45 to the Aspergillus, and 13 to the Penicillium groups. The location possessed a higher influence than farming method on the distribution of mould genera. Despite the requirement of higher temperature, Aspergillus species dominated only for the region of Újfehértó with approximately 50% of the isolates belonging to the genus. Four of the seven locations assessed: Csenger, Debrecen-Pallag, Nyírtass and Nagykálló, were dominated by Alternaria species. All isolates belonging to the genera Aspergillus and Penicillium were tested for the presence of the isoepoxidone dehydrogenase (idh) gene, a key player in the patulin metabolic pathway. To guarantee patulin production, this ability was confirmed with TLC assays. The only Aspergillus strain that presented a positive result was the strain Aspergillus clavatus B9/6, originated from the apple cultivar Golden Reinders grown in Debrecen-Pallag by integrated farming. Of the Penicillium isolates only one strain, B10/6, presented a band of the right size (500-600 bp) for the idh gene. Further sequencing of the ITS gene showed that this strain should be classified as Talaromyces pinophilus. The TLC tests confirmed this microorganism as the only patulin producer under the studied conditions for its cluster.

Glass bead system to study mycotoxin production of Aspergillus spp. on corn and rice starches.

Mycotoxin production by aflatoxin B1 (AFB1) -producing Aspergillus flavus Zt41 and sterigmatocystin (ST) -hyperproducer Aspergillus creber 2663 mold strains on corn and rice starch, both of high purity and nearly identical amylose-amylopectin composition, as the only source of carbon, was studied. Scanning electron microscopy revealed average starch particle sizes of 4.54 ± 0.635 µm and 10.9 ± 2.78 µm, corresponding to surface area to volume ratios of 127 1/µm for rice starch and 0.49 1/µm for corn starch. Thus, a 2.5-fold difference in particle size correlated to a larger, 259-fold difference in surface area. To allow starch, a water-absorbing powder, to be used as a sole food source for Aspergillus strains, a special glass bead system was applied. AFB1 production of A. flavus Zt41 was determined to be 437.6 ± 128.4 ng/g and 90.0 ± 44.8 ng/g on rice and corn starch, respectively, while corresponding ST production levels by A. creber 2663 were 72.8 ± 10.0 µg/g and 26.8 ± 11.6 µg/g, indicating 3-fivefold higher mycotoxin levels on rice starch than on corn starch as sole carbon and energy sources. KEY POINTS: • A glass bead system ensuring the flow of air when studying powders was developed. • AFB1 and ST production of A. flavus and A. creber on rice and corn starches were studied. • 3-fivefold higher mycotoxin levels on rice starch than on corn starch were detected.

Advantages of Multiplexing Ability of the Orbitrap Mass Analyzer in the Multi-Mycotoxin Analysis.

In routine measurements, the length of the analysis time and nfumber of samples analysed during a time unit are crucial parameters, which are especially important for the food analysis, particularly in the case of mycotoxin determinations. High-resolution equipment, including time-of-flight or Orbitrap analyzators, can provide stable instrumental background for high-throughput analyses. In this report, a short, 1 min MS-based multi-mycotoxin method was developed with the application of a short column as a reduced chromatographic separation, taking advantages of the multiplexing and high-resolution capability of the QExactive Orbitrap MS possessing sub-1 ppm mass accuracy. The performance of the method was evaluated regarding selectivity, LOD, LOQ, linearity, matrix effect, and recovery, and compared to a UHPLC-MS/MS method. The final multiplexing method was able to quantify 11 mycotoxins in defined ranges (aflatoxins (corn, 2.8-600 µg/kg; wheat, 1.5-350 µg/kg), deoxynivalenol (corn, 640-9600 µg/kg; wheat, 128-3500 µg/kg), fumonisins (corn, 20-1500 µg/kg; wheat, 30-3500 µg/kg), HT-2 (corn, 64-5200 µg/kg; wheat, 61-3500 µg/kg), T-2 (corn, 10-800 µg/kg; wheat, 4-250 µg/kg), ochratoxin (corn, 4.7-600 µg/kg; wheat, 1-1000 µg/kg), zearalenone (corn, 64-4800 µg/kg; wheat, 4-500 µg/kg)) within one minute in corn and wheat matrices at the MRL levels stated by the European Union.

Flavobacterium hungaricum sp. nov. a novel soil inhabitant, cellulolytic bacterium isolated from plough field.

A Gram-negative bacterial strain, named Kb82, was isolated from agricultural soil and a polyphasic approach was used for characterisation and to determine its taxonomic position. Based on 16S rRNA gene sequence analysis, the highest similarity was found with Flavobacterium artemisiae SYP-B1015 (98.2%). The highest ANI (83.3%) and dDDH (26.5%) values were found with Flavobacterium ginsenosidimutans THG 01 and Flavobacterium fluviale HYN0086T, respectively. The isolate is aerobic with rod-shaped cells, positive for catalase and negative for oxidase tests. The DNA G+C content is 34.7 mol%. The only isoprenoid quinone is menaquinone 6 (MK-6). The major fatty acids are iso-C15:0, summed feature 3 (C16:1 ω7c/C16:1 ω6c) and iso-C17:0 3OH. The major polar lipid is phosphatidylethanolamine. On the bases of phenotypic characteristics and analysis of 16S rRNA gene sequences, it is concluded that strain Kb82T represents a novel species in the Flavobacterium genus, for which the name Flavobacterium hungaricum sp. nov. is proposed. The type strain of the species is strain Kb82T (= LMG 31576T = NCAIM B.02635T).

Comparison Evaluation of the Biological Effects of Sterigmatocystin and Aflatoxin B1 Utilizing SOS-Chromotest and a Novel Zebrafish (Danio rerio) Embryo Microinjection Method.

Aflatoxin B1 (AFB1) is a potent mycotoxin and natural carcinogen. The primary producers of AFB1 are Aspergillus flavus and A. parasiticus. Sterigmatocystin (STC), another mycotoxin, shares its biosynthetic pathway with aflatoxins. While there are abundant data on the biological effects of AFB1, STC is not well characterised. According to published data, AFB1 is more harmful to biological systems than STC. It has been suggested that STC is about one-tenth as potent a mutagen as AFB1 as measured by the Ames test. In this research, the biological effects of S9 rat liver homogenate-activated and non-activated STC and AFB1 were compared using two different biomonitoring systems, SOS-Chromotest and a recently developed microinjection zebrafish embryo method. When comparing the treatments, activated STC caused the highest mortality and number of DNA strand breaks across all injected volumes. Based on the E. coli SOS-Chromotest, the two toxins exerted the same genotoxicities. Moreover, according to the newly developed zebrafish microinjection method, STC appeared more toxic than AFB1. The scarce information correlating AFB1 and STC toxicity suggests that AFB1 is a more potent genotoxin than STC. Our findings contradict this assumption and illustrate the need for more complex biomonitoring systems for mycotoxin risk assessment.

Sphingobacterium hungaricum sp. nov. a novel species on the borderline of the genus Sphingobacterium.

A Gram-reaction-negative bacterial strain, designated Kb22T, was isolated from agricultural soil and characterized using a polyphasic approach to determine its taxonomic position. On the basis of 16S rRNA gene sequence analysis, the strain shows highest similarity (94.39 %) to Sphingobacterium nematocida M-SX103T. The highest average nucleotide identity value (71.83 %) was found with Sphingobacterium composti T5-12T, and the highest amino acid identity value (66.65 %) was found with Sphingobacterium olei HAL-9T. Cells are aerobic, non-motile rods. The isolate was found to be positive for catalase and oxidase tests. The assembled genome of strain Kb22T has a total length of 4,06 Mb, the DNA G+C content is 38.1 mol%. The only isoprenoid quinone is menaquinone 7 (MK-7). The major fatty acids are iso-C15:0 (28.4%), summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH) (25.7 %) and iso-C17:0 3-OH (19.7 %). Based on phenotypic characteristics and phylogenetic results, it is concluded that strain Kb22T is a member of the genus Sphingobacterium and represents a novel species for which the name Sphingobacterium hungaricum sp. nov. is proposed. The type strain of the species is strain Kb22T (=LMG 31574T=NCAIM B.02638T).

Sphingobacterium pedocola sp. nov. a novel halotolerant bacterium isolated from agricultural soil.

A Gram-reaction-negative halotolerant bacterial strain, designated Ka21T, was isolated from agricultural soil and characterised using a polyphasic approach to determine its taxonomic position. On the basis of 16S rRNA gene sequence analysis, highest similarity was found with Sphingobacterium alkalisoli Y3L14T (96.72%). Cells were observed to be aerobic, non-motile rods. The isolate was found to be able to grow between 0 and 10% of NaCl concentration. The assembled genome of strain Ka21T has a total length of 5.2 Mb with a G + C content of 41.0 mol%. According to the genome analysis, Ka21T encodes several glycoside hydrolases that may play a role in the degradation of accumulated plant biomass in the soil. Based on phenotypic characteristics and phylogenetic analysis, it is concluded that strain Ka21T represents a novel species in the Sphingobacterium genus for which the name Sphingobacterium pedocola sp. nov. is proposed. The type strain of the species is strain Ka21T (= LMG 31575T = NCAIM B.02636T).

Cellvibrio polysaccharolyticus sp. nov., a cellulolytic bacterium isolated from agricultural soil.

A novel Gram-reaction-negative bacterial strain, designated Ka43T, was isolated from agricultural soil and characterised using a polyphasic approach to determine its taxonomic position. On the basis of 16S rRNA gene sequence analysis, the strain shows highest similarity (97.1 %) to Cellvibrio diazotrophicus E50T. Cells of strain Ka43T are aerobic, motile, short rods. The major fatty acids are summed feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2-OH), C18 : 1 ω7c and C16 : 0. The only isoprenoid quinone is Q-8. The polar lipid profile includes phosphatidylethanolamine, phosphatidylglycerol, four phospholipids, two lipids and an aminolipid. The assembled genome of strain Ka43T has a total length of 4.2 Mb and the DNA G+C content is 51.6 mol%. Based on phenotypic data, including chemotaxonomic characteristics and analysis of the 16S rRNA gene sequences, it was concluded that strain Ka43T represents a novel species in the genus Cellvibrio, for which the name Cellvibrio polysaccharolyticus sp. nov. is proposed. The type strain of the species is strain Ka43T (=LMG 31577T=NCAIM B.02637T).

Aflatoxin B1 and Sterigmatocystin Binding Potential of Non-Lactobacillus LAB Strains.

Research on the ability of lactic acid bacteria (LAB) to bind aflatoxin B1 (AFB1) has mostly been focusing on lactobacilli and bifidobacteria. In this study, the AFB1 binding capacities of 20 Enterococcus strains belonging to E. casseliflavus, E. faecalis, E. faecium, E. hirae, E. lactis, and E. mundtii, 24 Pediococcus strains belonging to species P. acidilactici, P. lolii, P. pentosaceus, and P. stilesii, one strain of Lactococcus formosensis and L.garviae, and 3 strains of Weissella soli were investigated in MRS broth at 37 °C at 0.2 µg/mL mycotoxin concentration. According to our results, among non-lactobacilli LAB, the genera with the best AFB1 binding abilities were genus Pediococcus, with a maximum binding percentage of 7.6% by P. acidilactici OR83, followed by genus Lactococcus. For AFB1 bio-detoxification purposes, beside lactobacilli, pediococci can also be chosen, but it is important to select a strain with better binding properties than the average value of its genus. Five Pediococcus strains have been selected to compare their sterigmatocystin (ST) binding abilities to AFB1 binding, and a 2-3-fold difference was obtained similar to previous findings for lactobacilli. The best strain was P. acidilactici OR83 with 18% ST binding capacity. This is the first report on ST binding capabilities of non-Lactobacillus LAB strains.

Aflatoxin B1 and Sterigmatocystin Binding Potential of Lactobacilli.

Due to global climate change, mould strains causing problems with their mycotoxin production in the tropical-subtropical climate zone have also appeared in countries belonging to the temperate zone. Biodetoxification of crops and raw materials for food and feed industries including the aflatoxin B1 (AFB1) binding abilities of lactobacilli is of growing interest. Despite the massive quantities of papers dealing with AFB1-binding of lactobacilli, there are no data for microbial binding of the structurally similar mycotoxin sterigmatocystin (ST). In addition, previous works focused on the detection of AFB1 in extracts, while in this case, analytical determination was necessary for the microbial biomass as well. To test binding capacities, a rapid instrumental analytical method using high-performance liquid chromatography was developed and applied for measurement of AFB1 and ST in the biomass of the cultured bacteria and its supernatant, containing the mycotoxin fraction bound by the bacteria and the fraction that remained unbound, respectively. For our AFB1 and ST adsorption studies, 80 strains of the genus Lactobacillus were selected. Broths containing 0.2 µg/mL AFB1and ST were inoculated with the Lactobacillus test strains. Before screening the strains for binding capacities, optimisation of the experiment parameters was carried out. Mycotoxin binding was detectable from a germ count of 107 cells/mL. By studying the incubation time of the cells with the mycotoxins needed for mycotoxin-binding, co-incubation for 10 min was found sufficient. The presence of mycotoxins did not affect the growth of bacterial strains. Three strains of L. plantarum had the best AFB1 adsorption capacities, binding nearly 10% of the mycotoxin present, and in the case of ST, the degree of binding was over 20%.

Effects of Single and Repeated Oral Doses of Ochratoxin A on the Lipid Peroxidation and Antioxidant Defense Systems in Mouse Kidneys.

Ochratoxin-A (OTA) is a carcinogenic and nephrotoxic mycotoxin, which may cause health problems in humans and animals, and it is a contaminant in foods and feeds. The purpose of the present study is to evaluate the effect of oral OTA exposure on the antioxidant defense and lipid peroxidation in the kidney. In vivo administration of OTA in CD1, male mice (1 or 10 mg/kg body weight in a single oral dose for 24 h and repeated daily oral dose for 72 h or repeated daily oral dose of 0.5 mg/kg bodyweight for 21 days) resulted in a significant elevation of OTA levels in blood plasma. Some histopathological alterations, transcriptional changes in the glutathione system, and oxidative stress response-related genes were also found. In the renal cortex, the activity of the glutathione-system-related enzymes and certain metabolites of the lipid peroxidation (conjugated dienes, trienes, and thiobarbituric reactive substances) also changed.

Effect of Sterigmatocystin or Aflatoxin Contaminated Feed on Lipid Peroxidation and Glutathione Redox System and Expression of Glutathione Redox System Regulatory Genes in Broiler Chicken.

Authors studied the effect of sterigmatocystin from infected corn (STC), purified sterigmatocystin (PSTC), and aflatoxin B1 from infected corn (AFB1) on lipid peroxidation and glutathione redox parameters, including the expression of their encoding genes in a sub-chronic (14 days) trial. A total of 144 three-week-old cockerels was divided into four experimental groups (n = 36 in each). Control feed was contaminated with STC or PSTC (1590 µg STC/kg or 1570.5 µg STC/kg feed), or with AFB1 (149.1 µg AFB1/kg feed). Six birds from each group were sampled at day 1, 2, 3, 7 and 14 of mycotoxin exposure. As parameters of lipid peroxidation, conjugated dienes (CD) and trienes (CT) were measured in the liver, while malondialdehyde (MDA) concentration was determined in blood plasma, red blood cell hemolysate and liver. Reduced glutathione (GSH) concentration and glutathione peroxidase (GPx) activity were determined in the same samples, and expression of glutathione peroxidase 4 (GPX4), glutathione synthetase (GSS) and glutathione reductase (GSR) genes was measured by RT-PCR in the liver. STC, PSTC or AFB1 caused a slight, but not significant, increase in CD and CT levels; however, in the case of MDA, no increase was found in the liver. Glutathione redox system was activated in the liver by AFB1, but less markedly by STC/PSTC. PSTC and AFB1 resulted in a higher expression of GPX4, while GSS expression was down-regulated by AFB1 on day 1, but up-regulated by STC on day 2 and by both mycotoxins on day 7. However, on day 14, GSS expression was down-regulated by PSTC. Expression of GSR was low on day 1 in AFB1 and PSTC groups, but later it was up-regulated by AFB1. The observed changes regarding gene expression strengthen the hypothesis that the mild oxidative stress, caused by the applied STC doses, activates the glutathione redox system of broiler chickens.

Proteomics Analysis of Thermoplasma Quinone Droplets.

A novel type of lipid droplet/lipoprotein (LD/LP) particle from Thermoplasma acidophilum has been identified recently, and based on biochemical evidences, it was named Thermoplasma Quinone Droplet (TaQD). The major components of TaQDs are menaquinones, and to some extent polar lipids, and the 153 amino acid long Ta0547 vitellogenin-N domain protein. In this paper, the aim is to identify TaQD proteome components with 1D-SDS-PAGE/LC-MS/MS and cross reference them with Edman degradation. TaQD samples isolated with three different purification methods-column chromatography, immunoprecipitation, and LD ultracentrifugation-are analyzed. Proteins Ta0093, Ta0182, Ta0337, Ta0437, Ta0438, Ta0547, and Ta1223a are identified as constituents of the TaQD proteome. The majority of these proteins is uncharacterized and has low molecular weight, and none of them is predicted to take part in lipid metabolism. Bioinformatics analyses does not predict any interaction between these proteins, however, there are indications of interactions with proteins taking part in lipid metabolism. Whether if TaQDs provide platform for lipid metabolism and the interactions between TaQD proteins and lipid metabolism proteins occur in the reality remain for further studies.

Aflatoxin B1 and Zearalenone-Detoxifying Profile of Rhodococcus Type Strains.

Aflatoxin B1 (AFB1) and zearalenone (ZON) are dangerous mycotoxins due to their carcinogenicity or oestrogenicity. To alleviate negative effects on humans and animals, successful detoxification tools are needed. The application of microorganisms to biodegrade mycotoxins can be an effective way in food and feed industry enhancing food safety. Several Rhodococcus strains are effective in the degradation of aromatic mycotoxins and their application in mycotoxin biodetoxification processes is a promising field of biotechnology. In this study, we investigated the AFB1 and ZON detoxification ability of 42 type strains of Rhodococcus species. Samples were analysed by high-performance liquid chromatograph equipped with fluorescence detector for mycotoxin concentration and SOS-chromotest was used for monitoring remaining genotoxicity. Out of the 42 Rhodococcus strains, 18 could eliminate more than 90% of the applied AFB1 and the genotoxicity was ceased by 15 strains in 72 h (R. imtechensis JCM 13270T, R. erythropolis JCM 3201T, R. tukisamuensis JCM 11308T, R. rhodnii JCM 3203T, R. aerolatus JCM 19485T, R. enclensis DSM 45688T, R. lactis DSM 45625T, R. trifolii DSM 45580T, R. qingshengii DSM 45222T, R. artemisiae DSM 45380T, R. baikonurensis DSM 44587T, R. globerulus JCM 7472T, R. kroppenstedtii JCM 13011T, R. pyridinivorans JCM 10940T, R. corynebacterioides JCM 3376T). In case of ZON, only R. percolatus JCM 10087T was able to degrade more than 90% of the compound and to reduce the oestrogenicity with 70%.

Xylanibacillus composti gen. nov., sp. nov., isolated from compost.

A novel Gram-stain-positive bacterial strain, designated as K13T, was isolated from compost and characterized using a polyphasic approach to determine its taxonomic position. On the basis of 16S rRNA gene sequence analysis, the strain showed highest similarity (93.8 %) to Paenibacillus nanensis MX2-3T. Cells of strain K13T were aerobic, motile rods. The major fatty acids were anteiso C15 : 0 (34.4 %), iso C16 : 0 (17.3 %) and C16 : 0 (10.0 %). The major menaquinone was MK-7, the polar lipid profile included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and an aminophospholipid. The DNA G+C content was 52.3 %. Based on phenotypic, including chemotaxonomic characteristics and analysis of the 16S rRNA gene sequences, it was concluded that strain K13T represents a novel genus, for which the name Xylanibacillus gen. nov., sp. nov. is proposed. The type species of the genus is Xylanibacillus composti, the type strain of which is strain K13T (=DSM 29793T=NCAIM B.02605T).

Micrococcoides hystricis gen. nov., sp. nov., a novel member of the family Micrococcaceae, phylum Actinobacteria.

A Gram-stain-positive bacterium, designated TSL3T, was isolated from faeces of a porcupine, Hystrix indica, from the Budapest Zoo and Botanical Garden, Hungary. On the basis of 16S rRNA gene sequence analysis, the strain is phylogenetically related to the family Micrococcaceae. The highest 16S rRNA gene sequence similarity was found with Micrococcus terreus V3M1T (96.50 %) followed by Arthrobacter humicola KV-653T (96.43 %). Cells of strain TSL3T were aerobic, non-motile and coccoid-shaped. The main fatty acids were anteiso-C15 : 0 (54.4 %), iso-C16 : 0 (18.2 %) and iso C15 : 0 (9.7 %). The major menaquinone was MK-7, and the polar lipid profile included phosphatidylglycerol, diphosphatidylglycerol, dimannosylglyceride, trimannosyldiacylglycerol, phosphatidylinositol, three unknown phospholipids and two unknown glycolipids. Strain TSL3T showed the peptidoglycan structure A4alpha l-Lys - Gly - l-Glu. The DNA G+C content of strain TSL3T was 58.4 mol%. Phenotypic and genotypic characterisation clearly showed that strain TSL3T could be differerentiated from the members of other genera in the family Micrococcaceae. According to these results, strain TSL3T represents a novel genus and species, for which the name Micrococcoides hystricis gen. nov., sp. nov. is proposed. The type strain is TSL3T (=DSM 29785T=NCAIM B. 02604T).

Lipoprotein-like particles in a prokaryote: quinone droplets of Thermoplasma acidophilum.

Cytosolic, globular droplets with an average diameter of 50 nm were observed in vitrified Thermoplasma acidophilum cells by means of cryo-electron tomography. These droplets were isolated by column chromatography and immunoprecipitation protein purification methods. Subsequent chemical and biochemical analyses identified lipid and protein components, respectively. Two major lipid components, comigrating menaquinones at the solvent front and the slower migrating Thermoplasma polar lipid U4, were detected by TLC experiments. The major protein component was identified as the 153 amino acid long Ta0547 vitellogenin-N domain protein. This domain has been found so far exclusively in large lipid transport proteins of vertebrates and non-vertebrates. Blast protein database homology searches with Ta0547 did not return any eukaryal hits; homologous sequences were found only in thermo-acidophilic archaeons. However, a profile-sequence domain search performed with the vitellogenin-N domain (PF01347) hmm-profile against the T. acidophilum proteome returned Ta0547 as hit. Electron microscopy appearance of isolated droplets resembled to lipoprotein particles. However, no (tetraether) lipid layer could be detected on the droplets surface, rather hydrophobic compounds of the electron dense lumen were surrounded by a denser discontinuous protein boundary. Based on described features, these particles qualify for a novel lipoprotein particle category, what we nominated Thermoplasma Quinone Droplet.

Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches.

Thermobifidas are thermotolerant, compost inhabiting actinomycetes which have complex polysaccharide hydrolyzing enzyme systems. The best characterized enzymes of these hydrolases are cellulases from T. fusca, while other important enzymes especially hemicellulases are not deeply explored. To fill this gap we cloned and investigated endomannanases from those reference strains of the Thermobifida genus, which have published data on other hydrolases (T. fusca TM51, T. alba CECT3323, T. cellulosilytica TB100T and T. halotolerans YIM90462T). Our phylogenetic analyses of 16S rDNA and endomannanase sequences revealed that T. alba CECT3323 is miss-classified; it belongs to the T. fusca species. The cloned and investigated endomannanases belong to the family of glycosyl hydrolases 5 (GH5), their size is around 50 kDa and they are modular enzymes. Their catalytic domains are extended by a C-terminal carbohydrate binding module (CBM) of type 2 with a 23-25 residues long interdomain linker region consisting of Pro, Thr and Glu/Asp rich repetitive tetrapeptide motifs. Their polypeptide chains exhibit high homology, interdomain sequence, which don't show homology to each other, but all of them are built up from 3-6 times repeated tetrapeptide motifs) (PTDP-Tc, TEEP-Tf, DPGT-Th). All of the heterologously expressed Man5A enzymes exhibited activity only on mannan. The pH optima of Man5A enzymes from T. halotolerans, T. cellulosilytica and T. fusca are slightly different (7.0, 7.5 and 8.0, respectively) while their temperature optima span within the range of 70-75°C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9-1.7mM of KM and 80-120 1/sec of turnover number. We detected great variability in heat stability at 70°C, which was influenced by the presence of Ca2+. The investigated endomannanases might be important subjects for studying the structure/function relation behind the heat stability and for industrial applications to hemicellulose degradation.

Enhancing recombinant protein solubility with ubiquitin-like small archeal modifying protein fusion partners.

A variety of protein expression tags with different biochemical properties has been used to enhance the yield and solubility of recombinant proteins. Ubiquitin, SUMO (small ubiquitin-like modifier) and prokaryotic ubiquitin like MoaD (molybdopterin synthase, small subunit) fusion tags are getting more popular because of their small size. In this paper we report on the use of ubiquitin-like small archaeal modifier proteins (SAMPs) as fusion tags since they proved to increase expression yield, stability and solubility in our experiments. Equally important, they did not co-purify with proteins of the expression host and there was information that their specific JAB1/MPN/Mov34 metalloenzyme (JAMM) protease can recognize the C-terminal VSGG sequence when SAMPs fused, either branched or linearly to target proteins, and cleave it specifically. SAMPs and JAMM proteases from Haloferax volcanii, Thermoplasma acidophilum, Methanococcoides burtonii and Nitrosopumilus maritimus were selected, cloned, expressed heterologously in Escherichia coli and tested as fusion tags and cleaving proteases, respectively. Investigated SAMPs enhanced protein expression and solubility on a wide scale. T. acidophilum SAMPs Ta0895 and Ta01019 were the best performing tags and their effect was comparable to the widely used maltose binding protein (MBP) and N utilization substance protein A (NusA) tags. Moreover, H. volcanii SAMP Hvo_2619 contribution was mediocre, whereas M. burtonii Mbur_1415 could not be expressed. Out of four investigated JAMM proteases, only Hvo_2505 could cleave fusion tags. Interestingly, it was found active not only on its own partner substrate Hvo_2619, but it also cleaved off Ta0895.

The detection and phylogenetic analysis of the alkane 1-monooxygenase gene of members of the genus Rhodococcus.

Naturally occurring and anthropogenic petroleum hydrocarbons are potential carbon sources for many bacteria. The AlkB-related alkane hydroxylases, which are integral membrane non-heme iron enzymes, play a key role in the microbial degradation of many of these hydrocarbons. Several members of the genus Rhodococcus are well-known alkane degraders and are known to harbor multiple alkB genes encoding for different alkane 1-monooxygenases. In the present study, 48 Rhodococcus strains, representing 35 species of the genus, were investigated to find out whether there was a dominant type of alkB gene widespread among species of the genus that could be used as a phylogenetic marker. Phylogenetic analysis of rhodococcal alkB gene sequences indicated that a certain type of alkB gene was present in almost every member of the genus Rhodococcus. These alkB genes were common in a unique nucleotide sequence stretch absent from other types of rhodococcal alkB genes that encoded a conserved amino acid motif: WLG(I/V/L)D(G/D)GL. The sequence identity of the targeted alkB gene in Rhodococcus ranged from 78.5 to 99.2% and showed higher nucleotide sequence variation at the inter-species level compared to the 16S rRNA gene (93.9-99.8%). The results indicated that the alkB gene type investigated might be applicable for: (i) differentiating closely related Rhodococcus species, (ii) properly assigning environmental isolates to existing Rhodococcus species, and finally (iii) assessing whether a new Rhodococcus isolate represents a novel species of the genus.